The principal objectives of this study are to elucidate the structural and metabolic functions of polyunsaturated fatty acids or phospholipids with particular reference to their modulation by ethanol. To this end, we investigated novel routes and sources of oxygenated metabolites of the major polyunsaturate of the brain, docosahexaenoic acid (22:6). Chromatographic, mass spectrometric, and extraction techniques were developed in order to characterize C22:6 products enzymatically formed by rat brain homogenate in vitro. We observed several mono-, di-, and trihydroxylated products which have not been previously described. These structural identifications were obtained after HPLC-thermospray MS in the positive ion mode and negative ion thermospray analysis of the pentafluorobenzyl derivatives of purified peaks. In combination with organic reactions such as hydrogenation and methoximation, trihydroxy-22:6 and -22:5, dihydroxy-22:5, and monohydroxy-22:6 peaks were assigned. The synthesis of these leukotriene-like products is stimulated by low concentrations of ethanol. Progress in analyzing the structural functions of unsaturated lipids was made by the development of rapid and efficient phospholipid molecular species analysis by HPLC-thermospray. Simple and information-rich fragmentation patterns, which readily yield head group and fatty acyl composition, were observed. This analytic technique has been successfully applied to complex biological mixtures for choline, ethanolamine, and serine phosphoglycerides.